Nonthermal atmospheric pressure plasma enhances mouse limb bud survival, growth, and elongation.

The enhanced differentiation of mesenchymal cells into chondrocytes or osteoblasts is of paramount importance in tissue engineering and regenerative therapies. A newly emerging body of evidence demonstrates that appendage regeneration is dependent on reactive oxygen species (ROS) production and signaling. Thus, we hypothesized that mesenchymal cell stimulation by nonthermal (NT)-plasma, which produces and induces ROS, would (1) promote skeletal cell differentiation and (2) limb autopod development. Stimulation with a single treatment of NT-plasma enhanced survival, growth, and elongation of mouse limb autopods in an in vitro organ culture system. Noticeable changes included enhanced development of digit length and definition of digit separation. These changes were coordinated with enhanced Wnt signaling in the distal apical epidermal ridge (AER) and presumptive joint regions. Autopod development continued to advance for approximately 144 h in culture, seemingly overcoming the negative culture environment usually observed in this in vitro system. Real-time quantitative polymerase chain reaction analysis confirmed the up-regulation of chondrogenic transcripts. Mechanistically, NT-plasma increased the number of ROS positive cells in the dorsal epithelium, mesenchyme, and the distal tip of each phalange behind the AER, determined using dihydrorhodamine. The importance of ROS production/signaling during development was further demonstrated by the stunting of digital outgrowth when anti-oxidants were applied. Results of this study show NT-plasma initiated and amplified ROS intracellular signaling to enhance development of the autopod. Parallels between development and regeneration suggest that the potential use of NT-plasma could extend to both tissue engineering and clinical applications to enhance fracture healing, trauma repair, and bone fusion

Household, community, sub-national and country-level predictors of primary cooking fuel switching in nine countries from the PURE study

Introduction. Switchingfrom polluting (e.g. wood, crop waste, coal)to clean (e.g. gas, electricity) cooking fuels can reduce household air pollution exposures and climate-forcing emissions.While studies have evaluated specific interventions and assessed fuel-switching in repeated cross-sectional surveys, the role of different multilevel factors in household fuel switching, outside of interventions and across diverse community settings, is not well understood. Methods.We examined longitudinal survey data from 24 172 households in 177 rural communities across nine countries within the Prospective Urban and Rural Epidemiology study.We assessed household-level primary cooking fuel switching during a median of 10 years offollow up (∼2005–2015).We used hierarchical logistic regression models to examine the relative importance of household, community, sub-national and national-level factors contributing to primary fuel switching. Results. One-half of study households(12 369)reported changing their primary cookingfuels between baseline andfollow up surveys. Of these, 61% (7582) switchedfrom polluting (wood, dung, agricultural waste, charcoal, coal, kerosene)to clean (gas, electricity)fuels, 26% (3109)switched between different polluting fuels, 10% (1164)switched from clean to polluting fuels and 3% (522)switched between different clean fuels

Inhibition of apoptosis signal-regulating kinase 1 alters the wound epidermis and enhances auricular cartilage regeneration.

Why regeneration does not occur in mammals remains elusive. In lower vertebrates, epimorphic regeneration of the limb is directed by the wound epidermis, which controls blastema formation to promote regrowth of the appendage. Herein, we report that knockout (KO) or inhibition of Apoptosis Signal-regulated Kinase-1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAP3K5), after full thickness ear punch in mice prolongs keratinocyte activation within the wound epidermis and promotes regeneration of auricular cartilage. Histological analysis showed the ASK1 KO ears displayed enhanced protein markers associated with blastema formation, hole closure and regeneration of auricular cartilage. At seven days after punch, the wound epidermis morphology was markedly different in the KO, showing a thickened stratum corneum with rounded cell morphology and a reduction of both the granular cell layer and decreased expression of filament aggregating protein. In addition, cytokeratin 6 was expressed in the stratum spinosum and granulosum. Topical application of inhibitors of ASK1 (NQDI-1), the upstream ASK1 activator, calcium activated mitogen kinase 2 (KN93), or the downstream target, c-Jun N-terminal kinase (SP600125) also resulted in enhanced regeneration; whereas inhibition of the other downstream target, the p38 α/β isoforms, (SB203580) had no effect. The results of this investigation indicate ASK1 inhibition prolongs keratinocyte and blastemal cell activation leading to ear regeneration

Skeletal cell differentiation is enhanced by atmospheric dielectric barrier discharge plasma treatment.

Enhancing chondrogenic and osteogenic differentiation is of paramount importance in providing effective regenerative therapies and improving the rate of fracture healing. This study investigated the potential of non-thermal atmospheric dielectric barrier discharge plasma (NT-plasma) to enhance chondrocyte and osteoblast proliferation and differentiation. Although the exact mechanism by which NT-plasma interacts with cells is undefined, it is known that during treatment the atmosphere is ionized generating extracellular reactive oxygen and nitrogen species (ROS and RNS) and an electric field. Appropriate NT-plasma conditions were determined using lactate-dehydrogenase release, flow cytometric live/dead assay, flow cytometric cell cycle analysis, and Western blots to evaluate DNA damage and mitochondrial integrity. We observed that specific NT-plasma conditions were required to prevent cell death, and that loss of pre-osteoblastic cell viability was dependent on intracellular ROS and RNS production. To further investigate the involvement of intracellular ROS, fluorescent intracellular dyes Mitosox (superoxide) and dihydrorhodamine (peroxide) were used to assess onset and duration after NT-plasma treatment. Both intracellular superoxide and peroxide were found to increase immediately post NT-plasma treatment. These increases were sustained for one hour but returned to control levels by 24 hr. Using the same treatment conditions, osteogenic differentiation by NT-plasma was assessed and compared to peroxide or osteogenic media containing β-glycerolphosphate. Although both NT-plasma and peroxide induced differentiation-specific gene expression, neither was as effective as the osteogenic media. However, treatment of cells with NT-plasma after 24 hr in osteogenic or chondrogenic media significantly enhanced differentiation as compared to differentiation media alone. The results of this study show that NT-plasma can selectively initiate and amplify ROS signaling to enhance differentiation, and suggest this technology could be used to enhance bone fusion and improve healing after skeletal injury

Inhibition of ASK1 or upstream activators of ASK1 enhance ear hole closure.

<p>A) Two-millimeter holes were punched in WT ears on day 0 and NQDI-1 (100μmol/L) in DMSO was added in the site of the ear hole every weekday, DMSO was added as control. Comparison of ear closure in WT mice with NQDI-1 (100μmol/L) in DMSO to WT mice with DMSO shows greater hole closure by area from day 0 to 32. B) Representative images of ear closure on D32 measured are shown for the two treatments. Average percent of areas closure are shown. Data are presented as mean ± SEM, NQDI-1 ears showed accelerated and greater amounts of wound closure compared to DMSO ear. NQDI-1 ear had a closure rate of 56.2± 2.7% by area on D14 after punch, which was more efficient than DMSO ear which closed 44.7± 3.1%. On the D32 after punch NQDI-1 ear hole closed 70.9±1.9% when DMSO ear closed 48.3±2.7%. C) Hematoxylin and eosin (H&E) and alcian blue staining of NQDI-1 and DMSO ears on D32 after punch is shown. DMSO ear shows very little new cartilage, sebaceous glands and a lot of connective tissues while NQDI-1 ear showed more new cartilage, hair follicles and sebaceous glands. D) Histology of wound epithelia shows NQDI-1 treatment slows terminal differentiation of epithelial keratinocytes at D7 after wounding, similarly to that observed in the KO. Black scale bar = 500 μm. E—G) Results of the addition of inhibitors for p38, JNK, and CAMKII on ear hole closure. t-test was used to calculate p values. Statistical significance is displayed as p < 0.05 (*), p < 0.01 (**), p < 0.001 (***).</p

Loss of ASK1 increases ear hole closure.

<p>A) Two-millimeter holes were punched in ears on day 0 and closure was photographed at days 0, 7, 14 and 32 for WT and KO mice. B) A magnetic ear clip flattened the ear for photographing. C) Representative images of ear closure and average percent of areas closure are shown on D14 and D32 measured for WT, HET, and KO. Data are presented as mean ± SEM, ASK1 KO mouse ears showed accelerated, and greater amounts of wound closure compared to HET and WT ears. D) ASK1 KO mouse ears had a closure rate of 64.7± 1.6% by area on D14 after punch, which was more efficient than HET and WT ears which closed 60.4± 1.9% and 38.2±2.9%. On the D32 after punch ASK1 KO mouse ear hole closed 82.3±1.3% when HET and WT ears closed 69.6±2.2% and 53.1±2.2%. E) The analysis of frequencies of ear punch closure on the D32 after punch in WT, HET and KO mice. Inset shows Western blot with protein levels for ASK1 and actin for each genotype. p < 0.05 (one asterisk/mark), p < 0.01 (two asterisks/mark), p < 0.001 (three asterisks/mark). KO Vs. WT (*); KO Vs. HET (†); HET Vs. WT(#).</p

Protein markers indicating cellular activities of blastema formation were more prevalent in the ASK1 KO.

<p>A) Sections were stained for H2AX immunofluorescence, positive green nuclear fluorescent dots (white arrows) were consistently observed in the ASK1 KO tissue. The percentage of H2AX-positive cells was determined by counting all the positive cells per total cells in blastemal area in 3 fields from 3 mice. Data are presented as mean ± SEM. B) Similar DAB immunohistochemistry was performed with antibodies to p21 and MMP-13 positively stained cells are shown at black arrows in high magnification inset. Percent of positive stained cells/ total cells was determined and graphed for WT, HET, and ASK1 KO mice. White scale bar = 100 μm, black scale bar = 500 μm in low mag image and 100 μm in high mag. inset.</p

Regeneration of cartilage and ear tissues.

<p>A) A diagram of the mouse ear shows placement of the ear punch and the localization of the regenerating tissues. The thick dashed line indicates where the tissue section below was obtained and the dotted line demarcates the original and newly regenerated tissue in the hematoxylin and eosin (H&E) and alcian blue staining of WT, HET and ASK1 KO ears on D32 after punch. In the WT ear very little new cartilage (darker blue) has regenerated as compared to the HET and ASK1 KO ear. B) A higher magnification image of the black box in A showing regenerated cartilage (white arrow) C) A higher magnification image of the yellow box in A highlighting the presence of sebaceous glands (HET and KO–black arrow) Scale bar = 500 μm.</p